2 replies to this topic

[Rakesh Sharma]

I had a bacterial culture(in LB media) grown for 16hrs. As it was late in the night, i stored them at 4 degrees and proceeded to mini-prep for isolation of my plasmid of interest the next morning.
I faced a problem. After pelleting down the bacterial culture, the pellet wasn't hard, as it used to be. It gave a hazy, hairy appearance. After discarding the supernatant, I added TE buffer to re-suspend the pellet, the pellet could not be re-suspended. It remained a pellet.

Can any1 explain what happened? Is it advisable to store bacterial cultures at 4 degrees, or is the problem due to other reasons?

[philman]

storage overnight should have been ok at 4 degrees, athough any longer than that should really be at -80, are you sure you didn't start the miniprep last night? hairy and non-resuspending sounds like the protein precipitation stap of miniprep to me, could you have forgotton you did that?

other than that, no idea, sounds strange to me, in general I think next time if you want to leave the culture overnight the usual recommendation is to spin it down to a pellet, discard supernatant then store at -80 I think.

[perneseblue]

I agree with philman. THere should be no problems keeping the culture overnight at 4 Celsius. However it is also the norm to spin down the culture and freeze the cell pellet.

"hazy, hairy appearance."
It could be that the culture has began to flocculate.

However it could also mean that the culture was contaminated. Does the culture smell different or has a different colour compared to a normal e coli culture.